1. Test the concentration of the DNA sample(s).
2. Pipet the following into a microfuge tube:
Linearized vector DNA
Insert DNA (at 3:1 molar excess over vector)
10x ligation buffer
T4 DNA Ligase
Rest of volume
3. Vortex and spin briefly to collect drops.
4. Incubate the mixture at 16 degree for 60-120 min.
5. Use the ligation mixture for transformation.
Thoroughly mix the 10x ligation buffer before use.
The optimal insert/vector molar ratio is 3:1.
To minimize recircularization of the cloning vector, dephosphorylate linearized plasmid DNA with Alkaline Phosphatase(CIAP) prior to ligation. Heats inactivate the phosphatase or remove from the mixture after the dephosphorylation step.
DNA purity is an important factor for successful ligation. Plasmids should be purified using a method that will ensure isolation of high quality DNA. Use only high quality agarose and fresh electrophoresis buffers for gel-purification of DNA fragments.